Project Summary/Abstract: The dynamics of cell signals are instructive features that guide cell and tissue behavior. Yet, many important pathways, like the Wnt/?-catenin pathway, lack probes to observe and dissect endogenous signal dynamics with precision in space and in time. The overall goal of this proposal is to develop novel molecular probes that can visualize and perturb endogenous signal dynamics, with a focus on the Wnt/?-catenin pathway. Our proposal is divided into two projects. In the first project, we will develop a novel fluorescent biosensor to enable direct visualization of Wnt signal dynamics. Existing Wnt reporters can be dim and require genomic engineering of the target cell, act on slow transcriptional timescales that can obscure the upstream pathway dynamics, and provide no spatial information about the input Wnt signal. Because Wnt stimulation requires the aggregation of pathway co-receptor LRP6, observation of LRP6 oligomerization could provide a spatiotemporally resolved readout of pathway activity. We thus envision a new class of biosensor that allows observation of the aggregation of intracellular proteins. Our reporter must meet two important design criteria. First, it must report on endogenous protein clustering to avoid overexpression of signaling proteins or genomic modifications. Second, because physiological protein aggregates are small and often below the diffraction limit of visible light, our reporter must ?visually amplify? endogenous clusters such that they are visible by conventional microscopy. We describe plans to develop, characterize, and apply such a reporter, called CluMPS. CluMPS visually magnifies small endogenous protein clusters through principles of protein phase separation. Using both experiments and simulations, we will characterize the ability of CluMPS to detect and amplify intracellular protein aggregates, using optogenetic clustering of GFP as a model analyte. We will then apply CluMPS to detect clusters of endogenous proteins known to form physiological aggregates. Finally, we will apply CluMPS to detect the clustering of endogenous Wnt receptor LRP6 in response to cellular Wnt stimulation, and we will validate ?LRP6-CluMPS activity in cell, tissue, and developmental models. The modularity of CluMPS will allow its adaptation to generate sensors of diverse signaling pathways and cell states. In the second project, we will engineer the first optogenetic tools to allow inhibition of endogenous signaling pathways with spatiotemporal precision. We will target inhibition of both Wnt/?-catenin signaling and, separately, Ras-Erk signaling. We will validate successful pathway inhibition in the context of cell culture models of cancer and patterning of in vitro intestinal organoids. Notably, all of our probes will be designed in a modular fashion and thus could be readily modified to observe or inhibit diverse targets of interest. Success in our work will result in a suite of new tools to observe, perturb, and understand the fundamental biochemical processes that underlie normal physiology and its breakdown in disease, in close alignment with the central mission of NIGMS.